As described in above priority patent application Ser. No. 10/390,438, an effective tetrapyrollic photosensitizer, e.g. HPPH (a chlorophyll-a derivative) was conjugated with Gd(III)-aminophenyl-DTPA, an imaging agent. In vivo reflection spectroscopy confirmed that tumor uptake of the HPPH-aminophenylDTPA Gd (III) conjugate was higher than that of HPPH alone in the radiation-induced fibrosarcoma (RIF) tumor of C3H mice. The subcutaneously-implanted Ward colon carcinoma in rats showed markedly increased MRI signal at twenty-four hours after intravenous injection of the conjugate. Both in vitro (RIF tumor cells) and in vivo (mice bearing RIF tumors) the conjugate produced significant efficacy. We have synthesized a molecule [two Gd (III) atoms per HPPH molecule] that also remained tumor-avid, PDT-active, and with improved MRI enhancing ability than the related mono-Gd(III) analog. Unfortunately, at the MRI dose (10 μmole/kg), these conjugates produced severe skin phototoxicity. However, replacing the hexyl-group of the pyropheophorbide-a with a PEG group, produced remarkable tumor enhancing at 8 hour postinjection, significant tumoricidal activity. The poor water-solubility problem of these conjugates was resolved by liposomal formulation.
For many years, in vivo imaging of human organs was largely dependent upon the intravenous administration of radioactive molecules for nuclear scanning or non-radioactive iodinated chemicals for radiography. However, over the last decade magnetic resonance imaging (MRI) has assumed a critical role in imaging. Unlike nuclear scanning, conventional radiography, or even computed tomography, MRI uses contrast enhancers (“contrast media”) that contain paramagnetic ions, particularly gadolinium [Gd(III)]. They are not themselves “seen” by the MRI scanner. Rather, they affect the water in body tissue so as to increase its “signal” when placed in a magnetic field. At present, three similar gadolinium(III)-derived MRI contrast agents have been approved for human clinical use in the United States, the bis-N-methylglucamine salt of Gd(III)diethylenetriaminepentaacetic acid (DTPA) (Magnavist), the bis-N-methylamide of Gd(III) DTPA (Omniscan), and the Gd(III) chelate of 20-(2-hydroxypropyl) derivative of 1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-1,4,7-tetraacetic acid (Prohance). All three of these agents are carboxylate containing, water-soluble complexes. After intravenous injection they result in a transient signal increase in the vascular space and penetrate the “leaky” capillary bed of many tumors, but are rapidly excreted through the kidneys by glomerular filtration. Although several liver-specific contrast media have also been created, other organs have not been successfully targeted, and no specific tumor-avid MRI contrast agent is available to date.
Signal Intensity in MRI:
Signal intensity in MRI stems largely from the local value of the longitudinal and transverse relaxation rates of water protons, 1/T1, and the transverse rate, 1/T2. Signal tends to increase with increasing 1/T1 and decrease with increasing 1/T2. Pulse sequences that emphasize changes in 1/T1 are referred to as “T1-weighted,” and the opposite is true for T2-weighted scans. Contrast agents increase both 1/T1 and 1/T2 to varying degrees, depending on their nature as well as the applied magnetic field. Agents like gadolinium (III) that increase 1/T1 and 1/T2 by roughly similar amounts are best visualized using T1-weighted images, because the percentage change in 1/T1 in tissue is much greater than that in 1/T2. The longitudinal and transverse relaxivity values, r1, and r2, refer to the amount of increase in 1/T1 and 1/T2, respectively, per milimole of agent (often given as per mM of Gd). T1 agents usually have r2/r1 ratios of 1-2.
Advances in MRI have strongly favored T1 agents and thus gadolinium(III). Faster scans with higher resolution require more rapid radio-frequency pulsing and are thus generally T1-weighted, because MR signal in each voxel becomes saturated. T1 agents relieve this saturation by restoring a good part of the longitudinal magnetization between pulses. At the same time, a good T1 agent would not significantly affect the bulk magnetic susceptibility of the tissue compartment in which it is localized, thus minimizing any inhomogeneities that can lead to image artifacts and/or decreased signal intensity.
The effect of these agents is to increase signal on T1-weighted images that are negatively affected by proton density. The effect on T2-weighed images is to decrease signal, but this effect is minimal, because most of the T2 signal comes from the influence of proton density. Signal Intensity for the Spin Echo Imaging is expressed as:S(TE,TR)=N(H)[1−2e−(TR−TE/2)/T1=e−TE/T1]e−TE/T2 
Conventional clinical MRI units produce static, cross sectional images. Newer “interventional MRI” units allow the operator to continuously image an organ while performing surgery or other manipulations.
Gd(III) is a logical choice for MRI contrast media because of its superior performance compared with other lanthanide ions. Dysprosium(III) and holmium(III) have larger magnetic moments than that of Gd(III), but the asymmetry of their electronic states leads to very rapid electron spin relaxation. The symmetric S-state of Gd(III) is a more hospitable environment for electron spins, leading to a much slower electronic relaxation rate. In the process that gives rise to relaxivity, water protons hardly feel the effects of ions like Dy(III), much like a leaf near the rapid wings of hummingbird; Gd(III) electrons, on the other hand, are more closely in tune with the proton's frequency.
A key biological factor that influences the selection of gadolinium compounds for human use is that its ligands like DTPA circulate and are excreted intact. The metal ion is “buried” in the chelation cage and will not bind to donor groups of proteins and enzymes. This in vivo stability markedly reduces the potential for toxicity from free gadolinium.
Tetrapyrrole-Based Compounds as MRI Agents:
The porphyrins and related tetrapyrrolic systems are among the most widely studied of all macrocyclic compounds. In fact, in one capacity or another these versatile molecules have influenced nearly all disciplines in chemistry. The concentration of certain porphyrins and related tetrapyrrolic or expanded porphyrin-type compounds is much higher in malignant tumors than in most normal tissues. A few years ago Sessler and coworkers discovered a new class of expanded porphyrins that is based on the Schiff base condensation between a diformyl-tripyrrane and an aromatic 1,2-diamine. This new class of expanded porphyrins has come to be known as the “texaphyrins”. Compared to the natural porphyrin system, the texaphyrins possess a larger core size and thus have the capability to form complexes with certain lanthanides, including gadolinium(III). Gd(III) texaphyrin is currently under phase I/II human clinical trials as a tumor-avid MRI contrast agent.
Some tetrapyrrole-based compounds are effective photosensitizers for cancer treatment by photodynamic therapy [PDT]. Although PDT is sometimes considered a novel, idiosyncratic therapy, it has in fact been effective in a wide variety of malignancies, including skin, lung, bladder, head and neck, breast, and esophagus. The precise mechanism(s) of PDT are unknown; however, in vitro studies suggest that singlet oxygen production is phototoxic when the photosensitizing agent encounters light. In vivo animal data suggest that tumor vasonecrosis may be the direct cause of tumor kill.
Effective PDT requires delivery of light to tumor that has absorbed a photosensitizer previously delivered by the systemic circulation after peripheral intravenous injection. Superficial visible lesions, or those that are endoscopically accessible—e.g., endobronchial or esophageal—are easily treated, but the vast majority of malignant lesions are too deep to be reached by light of the wavelength required to trigger singlet oxygen production in the current generation of photosensitizers. Although the technology to deliver therapeutic light to deep lesions via thin transmission fibers “capped” by a terminal diffuser is well-developed, a deep lesion is by definition not visible from the skin surface, and its uptake of a peripherally-injected photosensitizer is unknown; therefore, PDT of deep tumors thus far been impractical.
A relatively long-wavelength absorbing photosensitizer, the 3-(1-hexyloxy)ethyl derivative of pyropheophorbide-a 1 [HPPH], developed in our laboratory, is tumor-avid and currently in Phase VIII clinical trials at The Roswell Park Cancer Institute. We investigated this compound as a “vehicle” for delivering gadolinium complexes to tumor, with the goal of creating the first single compound that would function both as an MRI tumor-avid contrast medium and a photosensitizer for cancer therapy. [Gd(III) texaphrin is not a photosensitizer, because it does not produce singlet oxygen when exposed to light].